DNA Enrichment for Methylation Analysis

Hello all
I am doing my research on methylation profiling of various TSGs in hepatocellular carcinoma. The sample for me are the fine needle liver biopsies. These I guess are a little small and the DNA which is expected to come out of these might not be more than 2-3 ug. I have a little larger set of TSGs to profile and also there is the issue of repeat experiments etc so I want to increase the quantity of my DNA.
The point is that I want to include a DNA enrichment step in my work. I have a couple of protocols with me like "whole genome amplification". I want the experts here in this forum to comment on this method and also please let me know if there is any other technique I can employ to have larger quantity of DNA.

with such kits you need to test in your own hands to ensure there is no bias between methylated and unmethylated templats by comparing a control region before and after amplification!!!!

I have tried it and for our control region it seems to work fine, but I am always skeptical with the test regions we have looked at.

good luck

Hello, I am new in the forum and I have exactly the same problem with my sample, since its DNA concentration is far below the 1 ug recommended for bisulfite treatment (I use CpGenome from Chemicon). I started to consider the possibility of using a kit to amplify the DNA already treated, like the whole bisulfitome amplification kit from Qiagen. Is there another opinion regarding this issue? And by the way, this question goes to methylnick, what kind of control region do you use to test a proper amplification? Please HELP ME!! Loretta.

Hello, I am new in the forum and I have exactly the same problem with my sample, since its DNA concentration is far below the 1 ug recommended for bisulfite treatment (I use CpGenome from Chemicon). I started to consider the possibility of using a kit to amplify the DNA already treated, like the whole bisulfitome amplification kit from Qiagen. Is there another opinion regarding this issue? And by the way, this question goes to methylnick, what kind of control region do you use to test a proper amplification? Please HELP ME!! Loretta.

I've used the Applied Biosystems MethylSeqr conversion kit with some success. It only requires 300ng of DNA input so it might be an alternative to WGA. As far as control region I am interested in what methylnick has to say as well.Good luck.

what kind of control region do you use to test a proper amplification? Please HELP ME!!

Anything that you know works in the past for you, we have plenty of assays that we know work in our hands, are you able to source some assays from another lab already working on this stuff? I know with the HGS methyleasy kit there are primers included in the kit to test for conversion.

In terms of amplification bias post WGA, I would reccommend a known imprinted region where you would expect approximately 50% methylated to unmethylated alleles in your sample, any significant biases will skew to the methylated or unmethylated allele accordingly.

Good Luck!

All comments and communication are my own personal ones, and are not tied to any of my affiliations.